Method of producing peptides

专利摘要:
Synthetic peptides are extremely potent in stimulating the release of pituitary GH in mammals because they are the replicates of the native (hormone) releasing factor of the hypothalamus of the particular species, i.e. porcine, bovine, caprine and ovine. These peptides contain the following sequence: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-R13-Leu-Gly-Gln-Leu-Se r-Ala-Arg- Lys-Leu-Leu-Gin-Asp-Ile-Met-R28-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gl n-Gly-Ala- R41-Val-Arg-Leu wherein R13 is Val or Ile; R28 is Ser or Asn; and R41 is Arg or Lys. The peptide or a biologically active fragment thereof, or analogs thereof having well-known substitutions and/or additions, as well as nontoxic salts of any of the foregoing, may be administered to mammals and may be used diagnostically. The peptides are particularly useful in stimulating the release of GH so as to accelerate growth in warm-blooded non-human animals of the particular species and/or to improve aquiculture.
公开号:SU1423000A3
申请号:SU843790663
申请日:1984-08-27
公开日:1988-09-07
发明作者:Болен Питер；Эрнест Бразо Поль；Стефен Эш Фредерик；Чай-Ван Линг Николас；Бьюз Веренберг Вильям；Чарльз Луис Гуйллемин Роджер
申请人:Дзе Салк Институт Фор Биолоджикал Стадиз (Фирма)；
IPC主号:

专利说明:

The invention relates to a method for producing peptides — new biologically active compounds that can be used in veterinary medicine I,
The purpose of the invention is to increase the availability of compounds of peptide nature, of low toxicity and possessing the property to stimulate the release of growth hormone by the pituitary gland.

Example 1. Synthesis of pork
G | F (1-44) HE of the formula: N-gTir-Ala-Asp-Ala-Ile-Fe-Tgr-Asn--fer-Tir-Arg-Liz-Val-Lei-Gli-Gln-e-Ser -Ala-Arg-Lees-Leu-Leu-Gln-Asp - Le-Met-Sir-Arg-Gln-Glu-Gli-Gli-4rg-Asn-Gln-Glu-Gln-Gli-Ala-Arg-Val - Dr-Leu-OH was carried out stepwise, using a Beckman 990 peptide synthesizer on a chloromethylated resin, manufactured by Lab.Systems. lac, containing 0.9 mEq C1 / g. By addition of BOC-Leu to the resin, pg | and this is replaced by approximately 1 mol Leu per 1 g of resin. All growers that are used in this case are thoroughly degassed by sparging with an inert gas, or preferred with helium, in order to be sure that there is no oxygen, which can lead to undesirable sulfur in the methane residue.
After removing protection and neutralizing. The peptide chain is constructed step by step on the resin. The binding is exactly carried out according to the scheme in the table.
 For the coupling reaction, 1 mmol of a Boc-protected amino acid in methylene chloride is used on 1 g of resin plus 1 eq of 0.5 M dicyclohexyl carbodegyde (DCCI) in methylene chloride or 30% DMF in methylene chloride for 2 hours. When Apr associated, use a mixture of 10% methylene chloride. Benzyl (Bel) is used as a protective group for the hydroxyl side chain of Ser and Trr. 2-Chlorobenzyloxycarbosh (2C1-Z) is used as a protecting group of the Liz side chain. Tosil is used to protect the guanidino group Apr and the carboxyl group Glu or Asp is protected as bei. sily ether. The phenolic hydroxyl group Tyr is protected by 2,6-dichloro-Yenzil. At the end of the synthesis, a peptide resin of the following composition is obtained:
o
five
0
3
0
five
five
U
X4-Tyr (X2) -Ala-Asp (X) -La-Ile-Fe-Tgr (X4) -An-Ser (X5) -Tyr (X) -Arg- (Xg) -Liz (X ) -Val-Leu-Gli-Gln-Lei-Ser (X y) -Ala-Arg (X) -Liz (X 7) -Lpy-Ley-Gln-Asp (X) -Ile-Met-Ser (X5) -Arg- (X 5-) -Gl n-Gln-Gli-Glu (X,) -Apr (X e) - -Asn-Gli-Glu (X,) - Gln-Gli-Ala-Arg (Hy) - Val-Arg (Hb) -Le-X5,
where X, X „
- Wok
25b-dichlorobenzyl;
X is benzyl ether;
X4 - Bel;
Xj - Toe;
Xt, -. 2C1-Z;
Xg - -OgCH ,, - benzenepolystyrene resinous carrier.
After the termination of the residue, the Tire is SEMETHED with resin, the Boc group is removed with 45% trifluoroacetic acid in methylene chloride (TFA). For cleavage and deprotection of the remaining protected peptide-resin system, the peptide is treated with 1.5 ip of anisole, 0.25 mp methyl methylsulfide and 10 ml of hydrogen fluoride (HF) per 1 g peptide-resin at -20 ° C for half an hour and at O From half an hour. After removal of HF under high vacuum, the residue of the resin-peptide is washed alternately with dry diethyl ether and chloroform, and the peptide is then extracted with degassed 2N. aqueous acetic acid. By lyophilizing the acetic acid extract, a white fluffy material is obtained.
The cleaved, deprotected peptide is then dissolved in acetic acid and subjected to fine-grained gel filtration using a Sephadex G-50 filter.
The peptide is then further purified by CM-32 carboxymethylcellulose cation exchange chromatography (1j8 X 18 CMs - 50 ml) 5 with a concave gradient 5 effect caused by dropping 1 l of 0.4 M HH40A5 pH 6.5 into a mixing flask containing 400 ml of 0.01 M, pH 4.5, Final purification is carried out using distribution chromatography on Sephadex G-50 with fine-grained carrier (Pharmacia) with n-BOH:; E10H: pyridine: 0.2% n . (4: 1: 7) solvent system. Chromatographic fractions are carefully monitored by thin layer chromatography (TC) and only those fractions that
turned out to be substantially pure, taken away.
Pig peptide GRF (1-44) (, in 1% acetic acid) - 64.2 and 1, yield 395.2 mg (9.5 g of peptide resin and 395.2 mg of peptide were obtained from 6 g of resin).
The synthesis is repeated using MBGA resin to obtain the same peptide, with amidated C-edge.
Pig peptide GRF (1-44) OH: (in 1% acetic acid) - 66 ,, yield 704.4 mg (from 9. g of resin, 9.80 g of peptide resin was obtained).
PRI mme R 2. Synthesis of bovine GRF (1-44) of the formula N-Tir-Ala-Asp-Ala-Ile Fen-Tre-Asn-Ser-Tir-Arg-Lees-Val-Lei-Gly-Gln -Ley-Ser-Ala-Arg-Lees-Lei-Lei-Gli-Asp-Ile-Met-As n-Arg-Gln-Gln-Gli-Glu-Arg-Asn-Gln-Glu-Gln-Gli-Ala Liz-Val-Arg-Ley-UN ,. step by step using the Beckmann peptide synthesizer 990 and 6 g of MBGA resin. The reaction of the combination of Boc-Leu with the molecule of this resin is carried out for 2 hours in methylene chloride using a three-fold excess of Boc-Leu and OS as an activating agent, resulting in a replacement of approximately 0.2-0.6 mmol Lei for every 1 g of amide bound resin with amino groups of MBHA resin. All used solvents are carefully removed by bubbling with an inert gas, preferably helium, in order to ensure the absence of oxygen, which could cause undesired oxidation of sulfur in the methane residue.
After removal of the protecting groups and neutralization on the resin molecule, the peptide chain is constructed step by step. Combination, deprotection and neutralization operations are carried out as described in Example 1.
For the coupling reaction, 1 mmol of Boc-protected amino acid in methylene chloride is used for each 1 g of resin plus 1 eq of 0.5-M DCC1 in methylene chloride or 30% DMF in methylene chloride, the reaction time is 2 hours. In the arginine coupling reaction, a mixture of 10% DMF with methylene chloride. Gasoline is used as the group protecting the side hydroxyl group in the serine and threonine molecules. n side chain lys sha as
- m.
ten
20
25
X, X, - X4 -
X. X, 423000
protecting groups use 2-x-morbenzyloxycarbonyl. Tosyl is used as a protective group for the guanidine group of arginine, and the carboxyl groups of glutamic and aspartic acids are protected as BZl-efIua. The phenolic hydroxyl group of tyrosine is protected by 2,6-dichlorobenzyl. Upon completion of the synthesis, the next product is prepared. but:
X, -Tir (Xg) -Ala-Asp (X,) - Ala-Ile Fen-Tre (X4) -Amn-Ser (Hu) -Tir (X,) - Apr (Hb) -Liz (XT) - Val-Leu-Gly-Gln-Lei-15 Ser (X5 -) - Ala-Arg (X) -Liz (X7) -Lei-Lei-Gln-Asp (He) -Ile-Met-Asp-Asn-Arg ( X,) - Gln-Gln-Gli-Glu (X,) - Arg (Khb) -Asn-Gln-Glu (X,) - Gln-Gli-Ala-Liz (X) -Val-Arg- (X) - Leu-N-MBHA polystyrene polymer substrate, where X i - Vos
2,6-dichlorobenzyl;
benzyl ether /
BZ1;
Bzl;
Tos;
2C1-Z.
After the final combination of the tyrosine residue with the resin molecule, the Nose group is removed using 45% THF in methylene chloride to give 19.4 g of the peptide-resin system. In order to remove and remove the blocking groups from the protected peptide-resin system, 10 g of the latter is treated with 10 MP of anisole 2.5 MP of methyl ethyl sulphide and 90 ml of hydrogen fluoride at -20 ° C for half an hour and at a temperature of 0 C for half an hour. After removal of hydrogen fluoride in high vacuum, the resin-peptide residue is washed alternately with dry diethyl ether and chloroform, and then the peptide is extracted with degassed aqueous 2N. acetic acid solution. As a result of the freeze-drying of the acetic acid extract, a white, friable material is obtained.
Next, the peptide cleaved and free of protecting groups is dissolved in 30% acetic acid and filtered using Sephadex C-50 fine gel.
Then the peptide is subjected to further purification by cation-exchange chromatography using carboxymethylcellulose CM-32 (1.8 x 18 cm; layer volume 50 mp) using concave
thirty
35
40
45
50
55
gradient. which are created by BfiHiieM 1 l of 0.4 M solution of ammonium acetate with a pH value of 6.5 in a mixing flask, in which 400 ml of 0.01 M ammonium nitrogen solution at pH value is equal 4.5. Xc | continuous purification is carried out using distribution chromatography on a finely dispersed ps | Sephadex C-50 (Pharmacia) with: using a solvent mixture of n-butyl) nol with ethanol, pyridine and O, 2% n. acetic acid in the ratio of 4: 1:: 1j: 7. Chromatographic fractions are thoroughly checked by thin-layer liquid chromatographic analysis - 3OJM, combining only those fractions that are characterized by a substantial degree of purity.
 The output of bovine GRF (1-44) NH, 2dl mg (from 3 g of resin), N (, in acetic acid) -64,0nn1.
g Using chloromethylated gum, get bovine GRF (1-44) HE with 128 mg bbcd (from 1 g resin), Cot-lj, (CJ ™ 1, B acetic acid -64.0 + 1.
PRI me R 3. Synthesis of sheep GRF (1-44) NN "formula:
3;
N-Tir-Ala-Asp-Ala-11pe-Fe-Tr-Asn-Ser-Tir-Lrg-Liz-Ile-Lei-Gli-Gli-Lei-Ser-Ala-Arg-Liz-Lei-Lei-Glnn Asp-Ile-Met-Asp-Arg-Gln-Gln-Gli-Glu-Arg-Asn-Gln-Glu-Gln-Gli-Ala-Li-Val-Arg-Lei-Sh is conducted stepwise using a Beckman synthesizer 990 and MBHA resin, as mentioned in Example 2. At the end of the synthesis, a peptide resin of the following composition is obtained: Q
X, -Tyr (XJ) -Ala-Asp (X,) - Ala-леle-Fy-Tgr (X4) -An-Ser (X5) -Tyr (X) -Arg (Hb) -Liz (XY)-леle -Ley-Gly-Gln-Ley-Ser (Khr) - Ala-Arg (Khb) -Liz (X) -Lei-Ley-Gln-Asp
The peptide-resin systems are carried out as indicated in Example 2. The cleaved and deprotected peptide is dissolved g in 30% acetic acid, subjected to fine-grained gel filtration using Sephadex G-50, cation-exchange chromatography and finally distribution chromatography, as
10 was indicated in example 1.
 The yield of sheep GRF (1-44) NHj 197 mg (starting from 3 g of resin), N- (, in 1% acetic acid) -62 ,.
15 The synthesis is repeated using a chloromethylated resin to obtain the same peptide having a free acidic C-edge, following the procedure of pp. described in example 1.
20 Output of sheep GRF (1-44) OH 186 mg (starting from 1 g of resin). (C 1, in 1% acetic acid) -64 ,.
Biological tests of NIN synthesized peptides,
The effectiveness of these peptides in the release of growth hormone in in vitro assays was determined. The experiments were carried out with the pituitary cell. Used cultures include rat pituitary cells removed previously for 4 or 5 days. Incubation with the substance to be tested is carried out for 3-4 hours and aliquots of the culture medium are removed in such a way as to measure the content in the immunoreactive GH using a well resolvable radioimmunoassay. The results of these comparative tests show that in an equimolar ratio peptide:
I pork GRF (1-44) NHo, where
. Shaft; Rjg Cepj R4, Apr, has internal biological activity
(Xs) -Ile-Met-Asn-Arg (X,) - Gln-Gln-Gli-natural peptide RFGRG (1-44) NH2.
Glu (X,) - Arg (Xe) -Asn-Gln-Glu (X ,,) - Gln-Gli / sha-Lease (X7) -Val-Arg (X,) - Ley-X 8 where X, - Bock ,
X - 2,6-dichlorobenzyl,
X is a benzyl ester,
X - Bal;
Xj, Bel 5
X - Tos $
X - 2C1-Z,
X „- NH-MBfA resinous carrier
about
After completion of the synthesis, the residue j np is combined with the resin, Boc group / gives 45% of the TFA in.-Cleavage .i deprotection of the remaining protected
II bovine GRF (1-44) R,
NH
g
Where
R, Bal; R2B Asn; R4i - Liz, and NH.
III
sheep's GRF (1-44) NH, where R, j- Ile; Rjg- Lei; R41 Liz have an activity of 70% of the activity of the natural peptide of RFGRG (1-44) NH.
The methods relate to low toxic substances.
Tests of peptides in acid form have shown that they have similar biological activity.
The in vivo experiments confirmed that peptides obtained under the conditions of the described method can
I pork GRF (1-44) NHo, where
. Shaft; Rjg Cepj R4, Apr, has internal biological activity
natural peptide RFGRH (1-44) NH2.
II bovine GRF (1-44) R,
NH
g
Where
R, Bal; R2B Asn; R4i - Liz, and NH.
III
sheep's GRF (1-44) NH, where R, j- Ile; Rjg- Lei; R41 Liz have an activity of 70% of the activity of the natural peptide of RFGRG (1-44) NH.
The methods relate to low toxic substances.
Tests of peptides in acid form have shown that they have similar biological activity.
The in vivo experiments confirmed that peptides obtained under the conditions of the described method can
7 1
be categorized as low-toxic compounds.
The proposed method allows to obtain new biologically active compounds - close analogues in structure and action to natural compounds, but more accessible. Synthesis of such compounds allows peptides to be produced with an amino acid sequence of a growth hormone releasing factor, a composition characteristic of certain animal species.
Thus, the implementation of the proposed method makes available synthetic versions of natural hormones, which allows synthetic hormones corresponding to their species to be introduced into the organism of different animals to achieve specific goals.

权利要求:
Claims (1)
[1]
Invention Formula
The method of producing peptides of general formula I
H-TyR-Ala-Asp-Ala-lle-Phe-ThR-AsN-SeR-TyR-ARg-Lys-R ,, - Leu-Cly-GlN-Leu-SeR-Ala-ARg-Lys-Leu-Leu- GlN-Asp-Ile-Met-R, e-ARg-GlN-Gly-Glu-Glu-ARg-AsN-GlN-Gly-GlN-Gly-Ala-R-Val-ARg-Ley-Y
where Y is OH or NHj-, R ,, is Val or lie, 16 SeR or AsN; R4, - ARg or Lys,
50
five
0
five
) i8
About 1 liter of BOC-LOU with 1 g of ICA or chloromethylated resin in chloromethylene, HJII dimethylformamide, or their mixture was removed bos-protection is carried out and the remaining amino acids in the sequence corresponding to general formula I are added to the resulting Leii polymer, the compound of general formula II X, -TyR (X) -Ala-Asp- (Xj) -Ala-Ile-Phe-ThR (X4) -AsN-SeR (X5) -TyR (X) -ARg (X5) - Lys (X4) -R ,, - Leu-Gly-GlN-Leu-SeR (X4) - Ala-ARg (X) - Lys (Xg) -Leu-Leu-GlN-Asp (X,) - ile-Met-Rae (X4) -ARg (X) -GlN-GlN-Giy-Glu (X3) -ARg (X5.) - AsN- GlN-Glu (X,) - GlN-Gly-Ala R4, (X5 or X) - Val-ARg (X,) - Leu-X, where X, is Bos;
X is 2,6-dichlorobenzyl,
X - benzyl ether;
HD - benzyl,
X - tosil;
Xfi - chlorobenzyloxycarbonyl,
Hu - chloromethylated
resin, NNMBGA
the carrier polymer is unblocked and detached using trifluoroacetic acid in methylene chloride, followed by treatment of the resulting product with hydrogen fluoride in the presence of anisole, methyl ethyl sulfide, or their mixture at O - 20 ° C.
Priority featured:
08.29.83 with R ,, j-Val; SeR; R, - ARg.
10/12/83 when R ,, - Val; AsN; R4, - Lys.
02.03.84 when R ,, - lie; - R, 2g- AsN; R4, - Lys.
+ 5% 1,2-ethanedithiol in CHjCl
(1 time) 0.5
50% trifluoroacetic acid +
+ 5% 1,2-ethanedithiol in
(1 time) 20
CHjCl rinse (3 times) 0.5
CH.jOH washing (2 times) 0.5
 AsN; R4, - Lys.
 - R, 2g- AsN; R4, - Lys.

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US06/527,292|US4610976A|1983-08-29|1983-08-29|Porcine GRF|

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US06/541,167|US4585756A|1983-10-12|1983-10-12|Bovine GRF|

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US06/585,814|US4605643A|1984-03-02|1984-03-02|Ovine GRF|

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